The science of genetics has taken a keen interest in the identification of human individuals and genetic relationships between individuals. The genome of an individual is unique to that individual, and can be used for identification purposes, e.g., testing for paternity and/or forensic testing (e.g. to identify an individual in the context of post-mortem identification or in the criminal justice system). Procedures have been developed which are based on identification and characterization of changes in an individual's DNA, referred to as DNA polymorphisms, where such changes are due to nucleotide substitution, insertion, or deletion within the chains of DNAs.
In forensics, for example, there is an interest in polymorphisms for identification purposes. Techniques have been developed to compare homologous segments of DNA to determine if the segments are identical or if they differ in one or more nucleotides. Practical applications of these techniques relate to fields other than forensic medicine, for example, genetic disease diagnosis and human genome mapping.
The most accurate and informative way to compare DNA segments requires a method which provides the complete nucleotide sequence for each DNA segment. Particular techniques have been developed for determining actual sequences in order to study mutation in human genes. See, for example, Proc. Natl. Acad. Sci. U.S.A. 85, 544-548 (1988) and Nature 330, 384-386 (1987). However, because of the extensive amounts of time and high costs to determine, interpret, and compare sequence information, presently it is not practical to use extensive sequencing for compare more than just a few DNA segments.
A frequently used technique for screening for DNA polymorphisms arising from mutations consist of digesting the DNA strand with restriction endonucleases and analyzing the resulting fragments by means of Southern blots. See Am. J. Hum.Genet. p32, 314-331 (1980) or Sci. Am. 258, 40-48 (1988). Since mutations often occur randomly they may affect the recognition sequence of the endonuclease and preclude the enzymatic cleavage at that site. Restriction fragment length polymorphism mappings (RFLPS) are based on changes at the restriction site. They are accurate but not very informative (PIC>0.3). The major problem with RFLPs is the inability of a test to detect changes that do not affect cleavage with a restriction endonuclease. In addition, the methods used to detect RFLPs are very labor intensive and expensive, especially the techniques which includes Southern blot analysis.
Another technique for detecting specific mutations in particular DNA segment involves hybridizing DNA segments which are being analyzed with a complementary, labeled oligonucleotide probe. See Nucl. Acids Res. 9, 879-894 (1981). Since DNA duplexes containing even a single base pair mismatch exhibit high thermal instability, the differential melting temperature can be used to distinguish target DNAs that are perfectly complimentary to the probe from target DNAs that only differ by a single nucleotide. See, e.g., U.S. Pat. No. 4,683,194. Further, subtle genetic differences among related individuals regarding nucleotides which are substituted in the DNA chains are difficult to detect. VNTR's or Jeffrey's probes are very informative but labor intensive, in distinction to microsatellites which are equally informative PCR based tests.
Short tandem repeat (STR) polymorphisms are commonly used in DNA identification, either as adjuncts to other genetic tests, or as stand-alone tests. Typically, when STRs are used for human identification, they are amplified in groups of three to four loci (multiplex amplification). Generally, the resulting amplified fragments are analyzed by polyacrylamide gel electrophoresis. Polymorphisms are thus typed according to size by comparing to similarly labeled known external standards or differently labeled internal standards. U.S. Pat. No. 5,364,759 describes the genus of simple tandem repeats as well as a DNA typing method employing the simple tandem repeats and PCR amplification of the loci. Fragments are analyzed by differential labeling of the products.
A critical parameter in DNA typing is the power of exclusion for the system. Power of exclusion is the ability of a test to exclude a falsely accused individual based on the individual's genetic characteristics. The commonly used STR multiplexes have exclusion probabilities in the range of 85% to 91%. This compares unfavorably with restriction fragment length polymorphic loci (RFLP loci), which often provide an equivalent power with just one locus. STR testing batteries which include greater numbers of lower power systems are more susceptible to this problem than are RFLP testing batteries which include a smaller number of higher power systems. The low exclusion probabilities of commonly used STR loci are the most negative aspect of their use, although the frequencies of both alleles of an individual can be included in calculating match. Although it is simpler and faster to perform DNA typing with STR loci than with RFLP loci and it can be performed with much smaller quantities of DNA, typing using STR loci sacrifice in exclusion power. Another disadvantage of current STR multiplex DNA typing systems is that the amplification is rarely, if ever, clean. In other words there is considerable formation of spurious bands, which is thought to be due to DNA polymerase slippage and mis-priming events (see e.g., Tautz D., Hyper variability of Simple Sequences as a General Source for Polymorphic DNA Markers, Nuc. Acids Res., 17(16) 6463-70 (1989)).
Other polymorphisms take the form of single nucleotide variations between individuals of the same species. Such polymorphisms are far more frequent than RFLPS, STRs and VNTRs. Some single nucleotide polymorphisms occur in protein-coding sequences, in which case, one of the polymorphic forms may give rise to the expression of a defective or other variant protein and, potentially, a genetic disease. Other single nucleotide polymorphisms occur in noncoding regions. Some of these polymorphisms may also result in defective protein expression (e.g., as a result of defective splicing). Other single nucleotide polymorphisms have no phenotypic effects.
Single nucleotide polymorphisms (SNPs) can be used in the same manner as RFLPs, and VNTRs but offer several advantages. Single nucleotide polymorphisms occur with greater frequency and are spaced more uniformly throughout the genome than other forms of polymorphism. The greater frequency and uniformity of single nucleotide polymorphisms means that there is a greater probability that such a polymorphism will be found in close proximity to a genetic locus of interest than would be the case for other polymorphisms. Also, the different forms of characterized single nucleotide polymorphisms are often easier to distinguish than other types of polymorphism, e.g., by use of assays employing allele-specific hybridization probes or primers).
There is a need in the art for a very accurate genetic relationship test procYedure which uses very small amounts of an original DNA sample, yet produces very accurate results. This is particularly true in the forensic medicine area and criminology because often only very small samples of DNA available.